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Junb knockout in zebrafish phenocopies the MO-mediated Junb knockdown PL phenotype. (a) Left panel: Bright field images of representative embryos at 72 hpf injected with the indicated CRISPR gRNA: co-CRISPR (400 pg), junba-CRISPR (250 pg), junbb-CRISPR (250 pg), junba-CRISPR + junbb-CRISPR (200 pg + 200 pg). Middle panel: Confocal images of trunk vasculature in Tg(fli1:EGFP)y1 embryonic region marked by rectangle on the left. Right panel, enlarged view of trunk region marked by the yellow rectangle in the middle panel. DA, dorsal aorta; PCV, posterior cardinal vein; ISVs, intersegmental vessels; PL marked with yellow arrows or if absent with asterisks. Panels are lateral view; dorsal is up, anterior to the left. Scale bars represent 500 µm (black) and 50 µm (white). (b) Percentage of PL defects in embryos (at 48 and 72 hpf) injected with CRISPR gRNAs as described in (a) against either junba (n = 68), junbb (n = 94) or in combination against both (n = 150) versus embryos injected with control CRISPR at corresponding doses of 250 pg (n = 99) or 400 pg (co-CRISPR, n = 105). Sum of 16 hemisegments per embryo was categorized into groups with no, partial or complete absence of PLs. ***P < 0.001, Mann-Whitney U-test. (c) Quantification of arterial and venous ISVs (aISV, vISV) given as % aISV and vISV in Tg(fli1:EGFP)y1 zebrafish embryos at 72 hpf injected with indicated CRISPR gRNAs. n = 40 embryo/group, mean ± SD. **P < 0.01, ns, not significant, Student’s t-test.
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